Rac1 controls cell turnover and reversibility of the involution process in postpartum mammary glands

Cell turnover in adult tissues is essential for maintaining tissue homeostasis over a life span and for inducing the morphological changes associated with the reproductive cycle. However, the underlying mechanisms that coordinate the balance of cell death and proliferation remain unsolved. Using the mammary gland, we have discovered that Rac1 acts as a nexus to control cell turnover. Postlactational tissue regression is characterised by the death of milk secreting alveoli, but the process is reversible within the first 48 h if feeding recommences. In mice lacking epithelial Rac1, alveolar regression was delayed. This defect did not result from failed cell death but rather increased cell turnover. Fitter progenitor cells inappropriately divided, regenerating the alveoli, but cell death also concomitantly accelerated. We discovered that progenitor cell hyperproliferation was linked to nonautonomous effects of Rac1 deletion on the macrophageal niche with heightened inflammation. Moreover, loss of Rac1 impaired cell death with autophagy but switched the cell death route to apoptosis. Finally, mammary gland reversibility failed in the absence of Rac1 as the alveoli failed to recommence lactation upon resuckling.


Introduction
Cell turnover in adult tissues is characterised by the death of older cells and replacement with new through stem and progenitor cell proliferation. How these processes are balanced to maintain long-term tissue homeostasis is not clearly understood. The mammary gland is an example of a tissue that maintains a state of flux throughout the adult life. It also undergoes periods of profound growth and regression in each reproductive cycle, providing a tractable model to study cell turnover. The primary role of the mammary gland is to produce milk as a source of nutrients to feed the newborn. Successful lactation depends on the coordinated development and differentiation of the secretory alveolar epithelium during pregnancy and the subsequent removal of these milk-producing units once the milk supply is no longer a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 needed. The balance of cell death and cell division continuously alters to permit tissue growth and regression within the mammary gland reproductive cycle, but little is known about how this is coordinated.
Weaning of the infants triggers the mammary gland to enter postlactational involution, a process in which the surplus milk secreting alveolar epithelium is pruned from the ductal tree using a controlled cell death program [1]. In rodents, 90% of the alveolar epithelium laid down in pregnancy is subsequently removed during involution, with the balance of cell turnover tipped towards cell death and the remodelling process completed within approximately two weeks. In murine models of forced involution, simultaneous weaning of the pups at the peak of lactation causes the secretory alveoli to become engorged with milk as production continues for the first 24 h, after which they dedifferentiate. Both mechanical stretch and milk factors have been reported to stimulate cell death [1,2]. A number of programmed cell death mechanisms have been identified in postlactational involution, including apoptosis, lysosomal permeabilisation, and cell death with autophagy, although the significance of the different death pathways is unclear [3][4][5][6][7]. Moreover, lysosomal permeabilisation and autophagy may feed into an apoptotic death downstream. Involution occurs in two phases; In the first 48 h, cell death is triggered but the process of involution is reversible and the gland can reinitiate lactation once suckling resumes [3,8]. The second phase is irreversible and is characterised by destruction of the subtending basement membrane, extensive alveolar cell death, and repopulation of stromal adipocytes. The dead cells and residual milk are primarily removed by neighbouring live alveolar mammary epithelial cells (MECs) that act as phagocytes [9][10][11][12]. Engulfment of milk fat by the nonprofessional MECs triggers lysosomal permeabilisation through a stat-3-dependent mechanism, which ultimately kills the cells [12]. In the second phase, professional phagocytes from the immune system enter the gland to engulf the remaining dead cells and the tissue remodels back to a state closely resembling the nulliparous gland [13,14].
We previously showed that the Rac1 GTPase plays a crucial role in postlactational mammary gland remodelling [9]. Rac1 is central to the conversion of MECs into nonprofessional phagocytes for the removal of dead cells and surplus milk and in controlling the inflammatory signature. We now reveal further roles for Rac1 in controlling the involution process. We have discovered that Rac1 acts as a nexus, controlling both the rate and balance of cell death and progenitor cell division in involution. Without Rac1, cell turnover accelerates with consequences on mammary gland remodelling in the irreversible phase. Moreover, failure to redifferentiate blocks mammary gland reversibility in the first phase of involution. Rac1 therefore has multifaceted roles in orchestrating the involution process.

Delayed alveolar regression and repopulation of adipocytes in involuting Rac1−/− mammary glands
Mammary gland remodelling in involution is accompanied by alveolar regression and concomitant fat pad repopulation in the surrounding parenchyma. To investigate the role of Rac1 in tissue regression during involution, we examined mammary tissues from female mice triggered to involute through simultaneous weaning of the pups. The Rac1 gene was deleted specifically in luminal cells of the mammary gland using Rac1 fl/fl :LSLYFP:WAPiCre (Rac1−/−) conditional knockout mice previously generated [15]. Cre-negative Rac1 fl/fl :LSLYFP littermates were used as wildtype (WT) controls. Analysis of the WT mammary glands by histology and immunofluorescence staining of epithelial and adipocyte markers revealed significant lobular alveolar regression and adipocyte repopulation between involution days 2 to 4 (Figs 1A, 1C, 1E, 1G, 1I, 1J, 1K and S1). By contrast, alveoli in Rac1−/− glands remained distended with  (Figs 1B, 1D, 1F, 1H, 1K and S1). Four weeks postinvolution, the alveoli had completely regressed in both WT and Rac1−/− glands, confirming that lobular alveolar cell death had occurred in the absence of Rac1 (Fig 1L).

β1-integrin is not upstream of Rac1 in alveolar regression
In mammary gland tissue, β1-integrin functions upstream of Rac1 to regulate lactational differentiation, stem cell renewal, and cell cycle progression [9,[16][17][18][19]; we thus investigated whether β1-integrin was linked to alveolar regression in involution. The β1-integrin gene was deleted in luminal cells of the mammary gland using β1-integrin fl/fl :LSLYFP:WAPiCre conditional knockout mice (Fig 2A). Crucially, tissue analysis at involution days 2 and 4 revealed that loss of β1-integrin did not impair alveolar regression and adipocyte repopulation compared to WT mice of a Cre-negative genotype (β1-integrin fl/fl :LSLYFP; Fig 2B-2F). This indicates that Rac1's role in tissue regression is elicited through a distinct upstream signalling axis.

Rac1 ablation imbalances cell turnover rates in involution causing delayed alveolar regression
We first investigated whether there was an initial delay in cell death in Rac1−/− glands, which might explain the delay in alveolar regression. At involution day 4 where the delayed regression is most prominent, numerous cell corpses were evident in Rac1−/− glands both within the alveolar epithelium and within the lumen space (S2A Fig). We confirmed the dead cells with cleaved caspase-3 staining and that they were of luminal cell origin with the Rosa: LSL-YFP reporter gene, which is activated in response to WAPiCre-induced recombination (Fig 3A and 3D). Moreover, cell death was not delayed within the earlier reversible involution stage (days 1 and 2), but rather increased cell corpses were detected in Rac1−/− tissue lumens (Figs 3B, 3C, 3E, 3F, S2A and S2B). However, this might be a result of defective clearance by epithelial phagocytes as opposed to increased cell death in Rac1−/− glands [9]. Thus, the delay in alveolar regression in transgenic glands is not linked to impaired cell death.
Retention of milk within the lumens might contribute to the distended alveolar phenotype in Rac1−/− glands, since Rac1 is crucial for epithelial cell-directed engulfment of apoptotic cell corpses and residual milk [9]. However, given the extensive cell death detected in early involution, it was surprising that the alveoli remained intact. We thus investigated whether Rac1−/− alveoli were being maintained through cell renewal. Ki67 staining revealed almost no detectable proliferation in WT glands at involution day 4. In contrast, Rac1−/− mammary glands showed extensive proliferation within duct and alveolar luminal cells and within cells in the surrounding stromal/fat pad areas (Figs 3G, 3H, 3L and S2C). To confirm that proliferation was occurring within Rac1−/− luminal cells and not from cells that had escaped recombination, we scored proliferation in YFP-positive/Rac1−/− cells using the Rosa:LSL:YFP reporter gene (Fig 3I and 3M) Proliferation within the transgenic ducts continued at 4 weeks postinvolution; at this stage, however, most of the lobular alveoli had regressed (Figs 1L, 3K and 3P). Analysis of the phase I-reversible stage of involution (days 1 and 2) revealed no significant difference and very little proliferation at day 1. In contrast, at involution day 2, increased of n = 3 mice per group. �� P � 0.01. (J) Alveolar regression and adipocyte repopulation at involution day 4 were confirmed by immunofluorescence staining with WGA-488 (green) to detect epithelium and perilipin antibody (magenta) to detect adipocytes. Bar: 100 μm. (K) Quantification of alveolar regression and adipocyte repopulation. Error bars: +/− SEM of n = 3 mice per group. �� P � 0.01. (J) Carmine staining of whole-mounted WT and Rac1−/− mammary glands at 4 weeks postlactational involution show complete regression of alveoli. Note: Bloated ducts persist in Rac1−/− glands. Bar: 0.7 mm. The data underlying the graphs shown in this figure can be found in S1 Data.
https://doi.org/10.1371/journal.pbio.3001583.g001 proliferation was detected in Rac1−/− tissues, but this was mainly confined to cells within the surrounding stromal/fat pad areas. Taken together, these results suggest that the delayed alveolar regression in early involution in Rac1−/− glands is linked to increased compensatory cell proliferation within the irreversible phase and not delayed cell death within the reversible phase. Thus, Rac1 has a key role in balancing the rate of cell death and proliferation in involution by limiting the division progeny of progenitors. Without Rac1, alveolar progenitors divide unexpectedly in involution. The newly replaced cells, however, have a limited life span and succumb to death as evidenced by subsequent alveolar regression 4 weeks postweaning involution.

Loss of Rac1 elicits distinct proliferation responses within the first and second gestation
Rac1 has been linked to cell cycle progression in numerous cultured cells and in vivo tissues [19][20][21][22]. This is in complete contrast to our findings in vivo in the involuting mammary gland where loss of Rac1 induces proliferation. We thus examined the effects of Rac1 deletion on glandular development and proliferation within the first and second gestation. In the first gestation, histological analysis of mammary glands at pregnancy day 18 revealed slightly smaller alveoli in Rac1−/− glands, but the area occupied by adipocytes was not significantly different ( Fig 4A-4C). Immunostaining with Ki67 and BrdU incorporation at lactation day 2 in the first cycle revealed no significant difference in proliferation between WT and Rac1−/− glands ( Fig  4D-4G). Moreover, detection of the Rosa:LSL:YFP reporter gene revealed that recombination and thereby gene deletion was extensive and the proliferation was occurring within YFP-positive/Rac1−/− cells and not from WT cells that had escaped recombination ( Fig 4D).
In marked contrast to the involuting gland, within the second lactation cycle at day 2, there was a severe block in proliferation of luminal cells with concomitant reduced lobular alveolar development in both late pregnancy and early lactation stages (Figs 4H-4N and S3). Taken together, these data show that Rac1 deletion has no effect on proliferation within the first lactation, heightened proliferation in postlactational involution, and severely defective proliferation within the second lactation. The disparity in proliferation profiles suggests stage-specific cell autonomous and possible nonautonomous regulation by Rac1.

Increased proliferation in involuting Rac1−/− glands is linked to inflammatory signals
To explain the contrasting effect on cell proliferation within different stages of the mammary gland cycle, we sought to investigate possible cell nonautonomous effects of Rac1. We previously showed heightened inflammatory responses in Rac1−/− mammary glands in postlactational involution [9]. Heightened inflammation has been linked to altered epithelial proliferation and pathogenesis within numerous tissues including the gut and skin; we thus sought to investigate whether altered inflammatory responses without Rac1 were linked to the increased proliferation. To test this, we first examined for presence of inflammatory signals within in the gestational stage and in involution. F4/80-positive macrophages and the macrophage recruitment and polarising chemokines CCL2 and CCL7 previously identified in gene expression arrays were used as markers of inflammation [9]. In the first gestation, Rac1 deletion did not induce inflammatory signals (Fig 5A and 5D-5F), and this correlated with no bars: +/− SEM of n = 4 mice per group. NS (not significant). The data underlying the graphs shown in this figure can be found in S1 Data.
https://doi.org/10.1371/journal.pbio.3001583.g002 To remove possible impending signals from inflammatory cells, pure populations of mammary epithelia were isolated from pregnant Rac1 fl/fl CreER mice and the Rac1 gene was ablated in culture with 4-hydroxytamoxifen (4oht). At this stage, greater than 90% of the cells are alveolar in origin. Loss of Rac1 impaired proliferation in 3D organoids on a basement membrane matrix (Fig 5G-5I). Moreover, ductal cultures from either nulliparous Rac1 fl/fl CreER mammary glands with 4oht-inducible Rac1 gene deletion or wild-type glands treated with a Rac1 inhibitor failed to branch in response to FGF2 treatment ( Fig 5J, 5L, 5M, 5M', 5O and 5O'). In contrast, treatment of wild-type MECs with 4oht for the same period did not perturb branching morphogenesis over 7 to 8 days (Fig 5K, 5N and 5N').
To test if morphogens released by macrophages cause proliferation in MECs, primary organoids embedded in a BM-matrix were cultured in conditioned media from M0 or M2 polarised macrophages. EdU incorporation showed a small but significant increase in proliferation in conditioned media compared to control (Fig 5P and 5Q). Together, these data suggest that increased proliferation in involution in Rac1−/− glands is in part driven through secondary non-cell-autonomous effects of Rac1 deletion and is connected to heightened and sustained inflammatory responses. These findings reveal that Rac1 mediates a functionally important cross-talk between mammary gland cells and immune phagocytes within their microenvironmental niche.

Rac1 directs cell death with autophagy but not apoptosis or necrosis
As cell death and inflammation are intimately linked, we examined the effects of removing Rac1 on the cell death route in involution. Ultrastructural studies in days 2 and 4, involuting glands revealed numerous vacuolar structures in WT alveolar epithelium but not Rac1−/− transgenics (Figs 6A, 6G, 6M, 6O and S4). Further analysis revealed autophagosomes and lysosomes in the WT alveolar epithelium, suggestive of cell death with autophagy (Figs 6A-6C, 6M, 6N and S4). Moreover, we detected numerous phagosomes with engulfed milk proteins, milk lipid droplets, and dead cells (Fig 6D-6F), which supports our previous findings showing engulfment through phagocytic cups and macropinosomes in WT cells [9]. In contrast, Rac1−/− alveoli were completely void of both autophagosomes and phagosomes; instead, we detected either live cells in the epithelium or dead cells shed into the lumen with late apoptotic   (Fig 6G-6L, 6O and 6P). We further confirmed autophagy in WT glands by immunostaining for the essential autophagy-related LC3β protein, which appeared punctate and therefore indicative of translocation to the autophagosome membrane. In contrast, LC3β staining was diffuse in Rac1−/− transgenics confirming the absence of autophagosomes ( Fig  6Q and 6R). Moreover, immunoblotting with the LC3β antibody showed reduced LC3II in Rac1−/− glands (Fig 6S and 6T). Consistent with the ultrastructural studies, gene array analysis revealed down-regulation of genes associated with autophagosome and lysosomal pathways ( Fig 6U). Whole groups of lysosomal hydrolases, including proteases, glycosidases, sulfatases, DNases, and lipases, were down-regulated in Rac1−/− transgenics (S1 Table). Taken together, these data show that Rac1 is required to induce cell death with autophagy but without Rac1 cells can still die via apoptosis and necrosis. The presence of necrotic cells likely contributed to the heightened inflammatory responses in Rac1−/− glands.

Cell death is accelerated without Rac1
We next investigated whether Rac1 affects the rate at which cells transit through death and whether cells die directly through primary or secondary necrosis. To test this, primary cultures of WT and Rac1−/− cells were induced to undergo anoikis, a detachment-induced cell death. We chose this method of cell death for three reasons; first, to prevent dying cells from removal by neighbouring nonprofessional phagocytosis, as single cells suspended in media are spatially out of reach for phagocytic removal; second, to trigger an innate programmed cell death rather than chemical-induced; and third, anoikis likely occurs in Rac1−/− glands in vivo as we previously showed loss of Rac1 perturbs cell-ECM adhesion with increased shedding of cells [9]. Dying cells incorporated higher levels of propidium iodide (PI) in the absence of Rac1 compared to WT controls, suggesting cell death by necrosis or late-stage apoptosis (Fig 7A-7C).
To establish the proximal cell death route, we first examined for hallmarks of apoptosis as this process accompanies a series of well-defined biological steps. Both WT and Rac1−/− cell corpses displayed intact membranes with nuclear condensation, late-stage membrane blebbing, body fragmentation, and stained positive for cleaved caspase 3 indicative of an apoptotic cell death (Figs 3B, 3E and 7D-7G). Early-stage apoptosis is characterised by phosphatidylserine exposure to the outer membrane leaflet, and Annexin V is commonly used to detect this motif. Colabelling with Annexin V and PI in cells suspended for 1 h and 5 h revealed that approximately the same number of cells enter apoptosis with and without Rac1 (Annexin V only); however, by 5 h, significantly more Rac1−/− cells proceed to late-stage apoptosis/necrosis (Annexin V/ PI) with a concomitant reduction in numbers in early-stage apoptosis (Annexin V only; Fig 7H). Moreover, the numbers of viable cells declined following an 8-h  suspension in the absence of Rac1 indicating that cells had proceeded through death and disintegrated within this time frame compared to WT controls (Fig 7I and 7J).
As Annexin V can also bind necrotic cells with ruptured membranes, some of the cells in the AnnexinV/PI fraction may be a result of primary necrosis. To address whether necrotic cells occurred secondary to apoptosis as a result of defective phagocytosis or whether Rac1 loss triggered necrosis, we analysed the nuclei of necrotic cell corpses by electron microscopy. In Rac1−/− glands, the nuclei of ruptured cells were not condensed, suggesting they had not entered the apoptotic pathway first, but rather died through primary necrosis (Fig 7L). Organelle spillage and cell necrosis were also detected in Rac1−/− primary cultures (Fig 7N). In contrast, WT dead cells had intact membranes with nuclear condensation (Fig 7K and 7M). These studies reveal that Rac1 slows down the process of programmed cell death. Without Rac1, cells die either through primary necrosis or through apoptosis, but death proceeds more rapidly than in WT epithelia. Taken together, loss of Rac1 increases cell turnover rates in the involuting mammary gland through both increased progenitor proliferation and accelerated cell death.

Lactation fails to resume upon pup resuckling in involuting Rac1−/− glands
To determine the functional consequences of the phenotypic defects in Rac1−/− glands, we investigated the reversible phase of the involution process. Nursing WT and Rac1−/− dams were separated from the pups for 48 h to stimulate the first phase of involution and then reunited for a 24-h period. Resuckling in the WT mammary glands recommenced lactation as detected by an approximate 18-fold increase in casein 2 gene expression, and the alveolar epithelium resumed a lactation morphology (Fig 8A, 8C and 8G). In contrast, the Rac1−/− glands failed to lactate, and the involution morphology persisted with dead cell shedding into the lumen (Fig 8D-8F and 8G). While a small increase in milk protein gene expression was detected in resuckled Rac1−/− glands compared with the involuting Rac1−/−, the casein gene expression was 18-fold less than the WT fed gland. This compromise in milk protein expression is significantly greater than the 2-fold decrease detected in the first lactation cycle where Rac1−/− dams are still able to support pups [9]. Together, these data indicate that Rac1 is crucial for mammary gland reversibility in phase I of the involution process upon resuckling.

Discussion
Our study reveals that Rac1 acts as a central nexus in controlling the balance of cell death and proliferation within the mammary gland and is critical for mammary gland reversibility in early involution. The mammary gland removes approximately 90% of its tissue weight in postlactational involution with mass destruction of the milk-secreting alveolar units. This extensive regression can only be accomplished if the balance tips towards cell death with reduced proliferation. We have discovered that Rac1 is central to maintaining the suppression of proliferation in involution. Removal of Rac1 induces extensive proliferation within the involuting gland within the irreversible phase. We show the initial delay in alveolar regression and concomitant fat pad repopulation is not due to delayed cell death but rather a lack of milk engulfment [9] accompanied by compensatory cell proliferation within alveoli. However, the newly replaced cells in alveoli have a short life span, as the alveoli regressed by 4 weeks postweaning. A recent study reported that engulfed milk lipids are recycled to expand adipocytes [23]. Thus, the delay in adipocyte repopulation in Rac1−/− tissues might not be solely due to space constraints but also availability of engulfed lipids for recycling.
A previous study also reported a delay in Rac1−/− alveolar regression but attributed the effects to delayed cell death [24]. In contrast, our data show that cell death is not blocked in Rac1−/− alveoli in early involution as we have detected numerous cell corpses using both light and electron microscopy.
The involution process is accompanied by inflammatory cell influx to remove residual dead corpses by phagocytosis, increased matrix metalloproteinase activity, and extracellular matrix remodelling with subsequent release of various morphogens [13,25]. It is well established that proinflammatory signals invoke stem cell proliferation in several disease models including psoriasis and various cancers. Accumulating evidence shows that the involution microenvironment supports breast cancer growth in tumour mouse models and promotes postpartum breast cancer in women [25][26][27][28][29]. Of interest is a study showing sustained up-regulation of the chemokine CCL2 increased cancer susceptibility in a transgenic mouse model [30]. Loss of Rac1 increases proinflammatory signals in the mammary gland including the chemokines CCL2 and CCL7. We have made the important discovery that one mechanism by which the postpartum involuting mammary gland protects itself from inflammation-induced proliferation is through the Rac1 GTPase. Interestingly, in the skin, loss of Rac1 also causes stem cell release through activation of c-myc, although this study did not investigate inflammatory NES; normalised enrichment score. The data underlying the graphs shown in this figure can be found in S1 and S2 Data files and S1 Table. https://doi.org/10.1371/journal.pbio.3001583.g006 responses [31]. Whether Rac1 promotes or suppresses cell proliferation appears to be dependent on the microenvironmental context. Rac1 is linked to stem cell renewal and cell cycle progression in mammary epithelia and in numerous other models [17,[19][20][21][22]. We have now shown that Rac1 genetic deletion perturbs proliferation in purified epithelial organoid cultures void of impending inflammatory cells. In contrast, organoids exposed to inflammatory signals proliferate. This suggests both cell autonomous and nonautonomous regulation of proliferation by Rac1 depending on the environmental context. In addition to heightened inflammatory signals, stagnant milk in Rac1−/− mammary gland structures linked to the defective engulfment might cause stretch-induced proliferation. Indeed, ductal and alveolar bloating is severe in Rac1−/− glands because of defective clearance by MEC phagocytes [9]. Studies in the Drosophila wing disc show dying cells promote growth in their neighbours through the Wnt family member Wingless and Dpp proteins [32,33]. It would be interesting to identify whether similar autonomous regulation also occurs in the mammary gland.
Upstream of Rac1, β1-integrin has been linked to stem cell renewal, cell cycle progression, and lactational differentiation in mammary epithelia [16,18,19,34,35]. Here, we demonstrate that in involution, Rac1 controls alveolar regression independently of β1-integrin. This suggests distinct upstream wiring allows Rac1 to perform multifaceted roles within the mammary gland. The Rac guanine nucleotide exchange factors (GEFs) ELMO and Dock180 also show delayed alveolar regression in involution, but the receptor that activates these GEFs remains to be identified [24].
Our data show that alveolar epithelial cells die through distinct mechanisms with and without Rac1. Multiple cell death mechanisms have been reported in mammary gland involution, including autophagy and lysosomal leakiness linked to milk fat engulfment [4,5,36]. Executioner caspases 3, 6 are detected in cell corpses in the first 48 h suggesting activation of these pathways, although once ablated cell death can also proceed independently of these caspases [4]. We discovered that in the absence of Rac1, cell death with autophagy is impaired; milk phagocytosis is impaired [9] with a concomitant lack of detectable lysosomes by EM and down-regulation of several lysosomal genes. Despite these deficiencies, Rac1−/− cells still die through apoptosis and necrosis, and there is no delay in cell death. These alternate routes ensure a protective redundancy that enables cell death to proceed. Of interest is that in the autophagy defective Beclin 1−/− mammary glands, Rac1 activation is perturbed, which suggests a regulatory feedback loop [5].
We have discovered that, in addition to increased proliferation, cell transit through the death process is accelerated without Rac1, thereby Rac1 critically functions to limit cell turnover rates in involution. This suggests a mechanism by which existing cells resist cell death to allow reversibility. One such mechanism is autophagy, which might act as a survival mechanism in early involution instead of cell death. We have identified this as an important self-regulatory mechanism that enables mammary gland reversibility in involution. Despite the presence of live alveolar cells within Rac1−/− alveoli in the first 48 h, the dedifferentiated cells cannot lactate upon resuckling. The lactation defect is long term, as we have previously   [9]. Future studies will focus on how perturbing Rac1 alters the luminal stem/progenitor niche leading to defective alveolar lineages and long-term tissue malfunction in successive gestations.

Ethics statement
Experiments with mice were approved by the University of Sheffield AWERB (Animal Welfare and Ethics Review Board) and conducted under the Home Office Licences PPL 70/8351 and PPL 1836785 awarded to NA. Mice were housed and maintained according to the UK Home Office guidelines for animal research.

Mice
The Rac1 fl/fl : YFP;WAPiCre Tg/• and Rac1 fl/fl ;CreER mice were as previously described [15]. For the in vivo analysis, the WAPiCre promoter, which is activated mid-late pregnancy was used for Rac1 fl/fl gene deletion specifically in luminal MECs. Rosa:LSL:YFP reporter gene was used to detect Cre-induced recombination of flox alleles. Rac1 fl/fl: YFP littermates that lacked the Cre gene were used as WT controls. The genotypes of offspring were determined by PCR amplification of ear DNA as in [15]. Rac1 fl/fl :YFP;CreER mice were used for inducible deletion of the Rac1 gene in primary cultures. Female mice were mated between 8 and 12 weeks of age. β1-integrin fl/fl :YFP:WAPiCre mice were generated by crossing β1-integrin flfl mice; JAX #004605 [37] with WAPiCre:YFP mice previously described [9]. For involution studies, dams were allowed to nurse litters (normalised to 6 to 8 pups) for 7 to 10 days, and then pups were weaned to initiate involution. In the involution rescue experiments, breeding trios were set up with a male, an experimental female, and a WT surrogate female. Impregnated females were separated from the male, allowed to litter and nurse offspring jointly as above. Experimental dams were separated for 48 h to involute, while the surrogates continued feeding the pups. Litters were subsequently reunited with the experimental female for a period of 24 h prior to gland harvesting. AroundAU : PerPLOSstyle; numeralsarenotallowedatthebeginningofasenten 3 to 5 mice per group were analysed for each developmental stage. For some experiments, mice were injected with BrdU 100 mg/kg for 2 h before harvesting.
Whole mount analysis was performed by spreading inguinal mammary glands on polysine slides and stained with carmine alum as previously described [34]. Glands were imaged using a Nikon SMZ18 stereoscope. reversibility in involution. Rac1 permits cell death with autophagy, which enables reversibility within the first 48 h if suckling recommences. Without Rac1, early inflammation, a lack of autophagy and concomitant alternative programmed cell death, prevents redifferentiation of cells. At 96 h, Rac1 mediates remodelling of mammary gland tissue with alveolar shrinkage. Without Rac1, heightened inflammatory signals stimulate luminal cell proliferation and the alveoli remain distended. The data underlying the graphs shown in this figure can be found in S1 Data.
https://doi.org/10.1371/journal.pbio.3001583.g008 TEM Involuting mammary tissues were fixed with 4% formaldehyde + 2.5% glutaraldehyde in 0.1 M HEPES buffer (pH 7.2) for 1 h. Postfixed with 1% osmium tetroxide + 1.5% potassium ferrocynaide in 0.1 M cacodylate buffer (pH 7.2) for 1 h, in 1% thyocarbohydrazide in water for 20 min, in 2% osmium tetroxide in water for 30 min, followed by 1% uranyl acetate in water for overnight. The next day tissues were stained with Walton lead aspartate for 1 h at 60˚C degree, dehydrated in ethanol series infiltrated with TAAB 812 hard grade resin, and polymerised for 24 h at 60˚C degree. TEM sections were cut with Reichert Ultracut ultramicrotome and observed with FEI Tecnai 12 Biotwin microscope at 100 kV accelerating voltage. Images were taken with Gatan Orius SC1000 CCD camera.

RNA isolation and cDNA synthesis
RNA was isolated from the fourth inguinal mammary gland using Trifast reagent (Peqlab) according to manufacturer's instructions. ApproximatelyAU : PerPLOSstyle; numeralsarenotallowedatt 2 μg of RNA was used to prepare cDNA using the high-capacity RNA to cDNA kit (Invitrogen) according to manufacturer's instructions.

Primary cell culture and gene deletion
Primary MECs were harvested from either 15.5 to 17.5 day pregnant mice for alveolar cultures or 12-week-old virgin mice for ductal cultures and cultured as described in [38].

Macrophage-conditioned media
Murine RAW 264.7 cells were cultured in DMEM (Sigma) supplemented with 1% (v/v) penicillin-streptomycin and 10% foetal bovine serum to 80% confluency. M2 polarisation was induced by adding 20 ng/ml of recombinant mouse IL4 (Bio-Techne) and recombinant mouse IL13 (Bio-Techne) for 24 h. Macrophages were left untreated for M0. Polarisation media was washed off and replaced with serum-free medium to generate conditioned media for 24 h, after which it was collected and filtered through a 0.22-μM pore filter to remove cell debris. ApproximatelyAU : PerPLOSstyle; numeralsarenotallowedatthebeginningofasentence:Pleasecheckandc 200 μg/ml Insulin-Transferrin-Selenium (Gibco) and 1 μg/ml hydrocortisone (Sigma) were added to the media.
Primary mammary cells embedded in a BM-matrix were cultured with either unconditioned media or conditioned media from M0 or M2 macrophages. Organoids were treated with 10 μM EdU for 2 h prior to processing.

Anoikis assays and FACS
Apoptotic MECs (−/+4oht) were prepared in suspension culture for either 1 h or 5 h, in serumfree media (DMEM-F12 containing 2.5 μg/ml insulin). Cells were labelled with PI and/or Alexa Flour 647-Annexin V (Biolegend) according to manufacturer's instructions and analysed by flow cytometry or cytospun onto polysine slides, fixed and counterstained with Hoechst for micrographs. Some cytospun suspension cultures were stained with cleaved caspase-3 antibody.

Immunostaining
Expression and distribution of various proteins were visualised by indirect immunofluorescence. Cells were fixed for 10 min in PBS/4% (w/v) paraformaldehyde and permeabilised for 7 min using PBS/0.2% (v/v) Triton X100. Nonspecific sites were blocked with PBS/10% goat serum (1 h, RT) prior to incubation with antibodies diluted in PBS/5% goat serum (1 h, RT, each). EdU was detected using the Click-iT EdU Alexa Fluor 647 imaging kit (Life Technologies), and nuclei were stained using 4 μg/ml Hoechst 33258 (Sigma) for 5 min at RT. Cells were washed in PBS before mounting in prolong gold antifade (Molecular Probes). Images were collected on a Nikon A1 confocal or a Leica TCS SP5 AOBS inverted confocal as previously described [15]. Nonbiased cell counts were performed by concealing the identity of each slide.

Protein analysis
Proteins were extracted as in [16]. Equal amounts of proteins were used and equivalent loading assessed by referral to controls, such as Calnexin (Bioquote SPA-860). Primary antibodies used for immunoblotting are indicated in S2 Table. ImageJ was used to quantify bands.
Supporting information S1 Fig. S1 Fig related to Fig 1. Table. S1 Table related